PROJECT SUMMARY Nitrite, once thought to simply be an intermediate of nitric oxide (NO) synthesis, has emerged as an important mediator of redox control and a major storage form of NO produced in the vasculature. It has become clear that nitrite itself is involved in the regulation of vascular tone, especially in response to hypoxia. Assays to measure nitrite are employed across a broad range of clinical research areas including those in asthma, diabetic ulcers, inflammatory and infectious disease, and traumatic brain injury. Because of these ongoing areas as well as nitrite?s important role in the vasculature, assays to measure nitrite have far-reaching impact within the biomedical research community. Despite the widespread use of techniques to measure nitrite as both an indirect marker of NO production and a pool of NO generation, current techniques to measure this species require significant pre-analytical processing, are prone to interference from other species, are incompatible with many sample matrices, and suffer from limited dynamic range or inadequate limits of detection. A standardized, accurate, facile and commercially ready device to measure nitrite would enable important clinical research for this compound. Such a device could accelerate and enable new insights into nitrite both as a product and source of NO synthesis, and potentially lead to the discovery of new diagnostic applications where nitrite holds value. To address the significant pre-analytical processing common to existing nitrite measurement techniques, we have begun to develop an innovative, straightforward and inexpensive device for measuring nitrite in small volumes of biological fluids. The device integrates our core device?a microfluidic NO sensor?with an additional electrode that selectively converts nitrite to NO. In this format, nitrite is selectively converted to NO, and the gaseous NO product is detected electrochemically across an NO gas-permeable membrane. On-chip conversion of nitrite to NO gas, followed by subsequent measurement of evolved NO offers several advantages: (1) reduced interference from other common interfering species and matrices due to the selectivity of the membrane; (2) signal amplification with the potential for higher sensitivity and lower limits of detection relative to traditional electrochemical techniques that measure nitrite directly; and (3) the ability to account for (and thus remove) the background signal associated with each unique sample matrix through an additional working electrode that is isolated from the electrode used to convert nitrite to NO. Leveraging our core competencies in NO chemistry and measurement, we have assembled a talented team to develop a simple-to- use, inexpensive device that can measure nitrite in samples employed by the biomedical research community.